Factor VII (FVII) is a vitamin K dependent glycoprotein which in its activated form (FVIIa) takes part in the coagulation process activating the Factor X and the Factor IX in the presence of calcium and of tissue factor. FVII is secreted in form of a single peptide chain of 406 residues, with a molecular weight of about 50 kDa. The FVII contains four distinctive structural domains: the N-terminal γ-carboxylic domain (Gla), two “epidermal growth factor (EGF)-like” domains, and a serine protease domain. The activation of the FVII into FVIIa is characterized by the cleavage of the Arg152-Ile153 domain (Arginine 152-Isoleucine 153) linkage. The FVIIa is, therefore, a compound with a light chain of 152 aminoacids with a molecular weight of about 20 kDa, and with a heavy chain of 254 aminoacids with a molecular weight of about 30 kDa) linked one to another with a single disulfide bridge (Cys135-Cys262).
The plasma FVIIa contains several post-translational modifications: the first ten glutamic acids are γ-carboxylated, Asp63 is partially hydroxylated, Ser52 (Serine 52) and Ser60 (Serine 60) are O-glycosylated and carry the Glucose(Xylose)0-2 and Fucose patterns, respectively, Asn145 (Asparagine 145) and Asn322 (Asparagine 322) are N-glycosylated mainly by biantennary bisialylated complex structures.
FVII is used for the treatment of patients suffering from hemophilia, showing a deficiency of Factor VIII (type A hemophilia) or of Factor IX (type B hemophilia), as of patients showing other deficiencies of coagulation factors, for example, a congenital deficiency of FVII. It is, therefore, necessary that concentrates of injectable FVIIa be available.
The most ancient method for obtaining FVIIa concentrates consisted of the purification of FVIIa from plasma proteins resulting from the fractionation.
For that purpose, the document EP 0 346 241 describes the preparation of a FVIIa-enriched fraction, obtained after adsorption then elution of a secondary product of the fractionation of plasma proteins containing the FVII and the FVIIa and other proteins such as Factors IX (FIX), X (FX) and II (FII), particularly the pre-eluate of PPSB (P=prothrombin or FII, P=proconvertin or FVII, S=Stuart Factor or FX and B=antihemophilic Factor B or FIX). The drawback of this process is that the obtained FVII still contains some traces of the other coagulation factors.
Likewise, the document EP 0 547 932 describes a manufacturing process of a high purity FVIIa concentrate substantially free of vitamin-K-dependent factors and of FVIII. The FVII obtained by this process, in spite of its purity, shows a residual thrombogenic activity.
Thus, one of major drawbacks of these processes is that they yield only small amounts of products. It is, moreover, still difficult to obtain a product entirely free of other proteins present in the plasma. Finally, although a number of precautions are being implemented at every stage of the preparation of plasma coagulation factors in order to ensure their viral and bacterial safety (follow-up of blood donors, tests for detecting of known viral and bacterial contaminants, stringent purification and virus inactivating treatments in order to reduce as far as possible the hazard of transmission of blood-born pathogenic agents), nevertheless, all risks of contamination with pathogenic agents are not excluded. In addition, the appearance of a new variant of the Creutzfeldt-Jakob disease gave rise to fears of transmission of unconventional pathogenic agents by blood products. Moreover, the volume of plasma collected from donors remains limited.
Therefore, since the 1980s, the DNA encoding the human Factor VII was isolated (Hagen et al. (1986); Proc. Natl. Acad. Sci. USA; April 83(8):2412-6) and expressed in mammal BHK cells (Baby Hamster Kidney) (document EP 0 200 421). Even if this method of manufacturing of FVII has the advantage to control the medium where the protein of interest is produced, it is known that the hamster cells impart to proteins which they express Galα1,3Gal moieties (Spiro R G et al, J. Biol. Chem., 1984, vol. 259, N° 15, 9858 and Furukawa K. et al., J. Biol. Chem., 1992, vol. 267, N° 12, 8012), the immunogenicity of which has been demonstrated.
It was found that 1% of the circulating human B lymphocytes raise antibodies against the epitope Galα1,3Gal (Galili et al, Blood, 1993, vol. 82, 2485). The epitope and the antibodies form then a complex activating the complement and leading to severe immunity reactions, such as acute transplant rejection following to xenotransplantations. It was shown that 15 to 20% of the hemophiliacs treated with a FVII produced in a hamster cell develop an immune reaction (Prowse C. V et al, Blood Reviews, 1998, vol. 12, 99). This type of immune reaction is tragic in the case of hemophiliacs, because the FVII and FVIII, having turned immunogenic, will cause bleedings which are very difficult to treat.